NLR Members in Inflammation-Associated

RESEARCH HIGHTLIGHT

NLR members in inﬂammation-associated carcinogenesis

Ha Zhu1,2 and Xuetao Cao1,2,3

Cellular & Molecular Immunology (2017) 14, 403–405; doi:10.1038/cmi.2017.14; published online 3 April 2017

hronic inflammation is regarded as an impor- nucleotide-binding and oligomerization domain IL-2,8 and NAIP was found to regulate the STAT3 Ctant factor in cancer progression. In addition (NOD)-like receptors (NLRs). TLRs and CLRs are pathway independent of inflammasome formation.9 to the immune surveillance function in the early located in the plasma membranes, whereas RLRs, The AOM/DSS model is the most popular model stage of tumorigenesis, inflammation is also known ALRs and NLRs are intracellular PRRs.3 Unlike used to study the function of NLRs in fl fl as one of the hallmarks of cancer and can supply other families that have been shown to bind their in ammation-associated carcinogenesis. In amma- the tumor microenvironment with bioactive mole- specific cognate ligands, the distinct ligands for somes initiated by NLRs or AIM2 have been widely cules and favor the development of other hallmarks NLRs are still unknown. In fact, mounting evidence reported to participate in the maintenance of 10,11 Nlrp3 Nlrp6 of cancer, such as genetic instability and angiogen- suggests that NLRs function as cytoplasmic sensors intestinal homeostasis. -/-, -/-, Nlrc4 Nlrp1 Nlrx1 Nlrp12 esis. Moreover, inflammation contributes to the and participate in modulating TLR, RLR and CLR -/-, -/-, -/- and -/- mice are 4 more susceptible to AOM/DSS-induced colorectal changing tumor microenvironment by altering signaling pathways. NLRs can sense stimulatory cancer than wild-type (WT) mice.11 Loss of IL-18 stromal cell turnover rates and polarizing immune signals from pathogens, host cells and environmen- may partially account for the enhanced tumorigen- cell immunosuppressive capabilities.1 In addition, tal sources, although the interaction patterns are 3 esis in the abovementioned mice. IL-18 production many tumor suppressor genes have been reported still unclear. Importantly, dysfunction of NLRs fl is mainly regulated by inflammasomes and can to be involved in inflammation modulation.2 How- leads to various in ammatory diseases, autoim- fl stimulate regeneration and repair in the colon.4 ever, the types of molecules and the underlying mune diseases and in ammation-associated fl carcinogenesis.5 Given the vital roles of NLRs in Molecules that can regulate the functions of the mechanisms involved in in ammation-associated fl inflammation and disease progression, research is in ammasome are currently gaining attention and cancer need to be further investigated, which may fi now focused on elucidating their roles in the are potential bene cial treatments for provide new targets for the control of cancer. inflammasome-associated disorders.12 Moreover, pathogenesis of diseases such as cancer, with the Recognition of danger-associated molecular pat- deficiency of NLRX1, NOD1 or NOD2, which are aim of finding new therapeutic targets. terns (DAMPs) and pathogen-associated molecular non-inflammasome NLRs, results in increased NLRs contain three conserved functional patterns (PAMPs) by pattern recognition receptors susceptibility to colorectal cancer. NLRX1 sensitizes fl domains: a C-terminal leucine-rich repeat for (PRRs) is the key step that modulates in amma- cells to apoptotic cell death in response to DSS ligand recognition, a central NACHT domain tion. Five major PRR families have been reported to treatment and promotes epithelial proliferation. essential for oligomerization and an N-terminal be involved in tumorigenesis, including the Toll- NLRX1 is a negative regulator of AKT and NF- protein-protein interaction domain. According to like receptors (TLRs), C-type lectin receptors κB. NOD1 participates in the maintenance of the the types of N-terminal domains, mammalian (CLRs), RIG-I-like receptors (RLRs), absent-in- intestinal epithelial barrier permeability and bal- NLRs are classified into NLRA, NLRB, NLRC and melanoma (AIM)-like receptors (ALRs) and the ances inflammatory responses under AOM/DSS NLRP subfamilies with acidic transactivation stimulation, whereas NOD2 regulates apoptosis domains, baculoviral inhibitory repeat (BIR)-like and the NF-κB and MAPK signaling pathways.11 1 Institute of Immunology, Zhejiang University domains, caspase recruitment domains and pyrin The functions of other non-inflammasome NLRs in 4,6 fi School of Medicine, Hangzhou 310058, China; domains. Since the rst NLR was discovered in inflammation-associated carcinogenesis are 2National Key Laboratory of Medical Immunology 7 early 2000, at least 23 distinct NLR and NLR-like unknown. Recently, Dr. Rajendra Karki’sresearch & Institute of Immunology, Second Military Med- fi 4 proteins have been identi ed in humans. NLRs, group published a paper in Nature that described ical University, Shanghai 200433, China and including NLRP1, NLRP3, NLRP6, NLPR7, 3 the function of a poorly researched non- Department of Immunology & Center for Immu- NLRP12, NLRC4 and NAIP, can function in an fl notherapy, Institute of Basic Medical Sciences, in ammasome-forming NLR, NLRC3, in colitis- inflammasome-dependent manner to produce 13 Peking Union Medical College, Chinese Academy associated carcinogenesis. interleukin-1β (IL-1β), IL-18 and caspase-1, thus NLRC3 was first identified in 2005 and found to of Medical Sciences, Beijing 100005, China fl Correspondence: Professor X Cao, National Key inducing in ammation and regulating cell pyrop- be highly expressed in T lymphocytes. NLRC3 has Laboratory of Medical Immunology and Institute tosis. By contrast, other NLRs such NLRP10, been reported to suppress the signaling path- of Immunology, Second Military Medical Univer- NOD1, NOD2, NLRC3, NLRC5, NLRX1 and ways involved in T cell activation by influencing sity, 800 Xiang Yin Road, Shanghai 200433, CIITA function independent of the inflammasome the transcription factors NF-κB, NFAT and China. and can regulate NF-κB and MAPK pathways or act AP1.14 When stimulated by virus infection, NLRC3 E-mail: [email protected] as transcriptional regulators.6 Interestingly, the negatively regulates the STING pathway in bone Received: 6 February 2017; Accepted: 6 February functional patterns of NLRs are not fixed. Recently, marrow-derived macrophages by directly associat- 2017 NLRP3 has been reported to regulate STAT5 and ing with STING and blocking STING traffic- NLR members in carcinogenesis H Zhu and X Cao

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king to puncta, thus inhibiting interferon-β expression.15 Reports on the functions of NLRC3 in carcinogenesis are rare. Karki et al.13 demonstrated that NLRC3 expression is significantly reduced in colorectal cancer tissues in mice; then, the authors established Nlrc3-/- mice and treated them with AOM/DSS. As expected, the Nlrc3-/- mice were more susceptible to the induction of colorectal cancer and exhibited greater weight loss, more tumor nodules, larger tumor sizes and higher histological scores. These findings indicate that NLRC3 acts as a suppressor in the development of colorectal cancer. Establishment of an inflammatory state is crucial for colitis-associated carcinogenesis.16 To assess the inflammatory state of the Nlrc3-/- mice treated with AOM/DSS, Karki et al.13 examined the expression of inflammatory cytokines, inflammatory signaling pathways and immune cell infiltration in colon tissues at days 80 and 14, which was 3 days after the first round of DSS treatment. There were no significant differences at day 80 between the Nlrc3-/- mice and WT mice. At day 14, pro- inflammatory cytokines, including IL-1β,IL-6, tumor necrosis factor, granulocyte colony stimulat- ing factor, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α,weregreatly increased in the Nlrc3-/- mice, whereas IL-18 remained unchanged. The NF-κB and STAT3 signaling pathways were activated at day 14, whereas the ERK signal pathway was unaltered. Thenumberofinnateimmunecells,including macrophages, neutrophils and natural killer cells, increased, whereas the numbers of B cells and T cells remained unchanged, and the number of dendritic cells decreased. These data demonstrate the hyper-inflammatory state of the Nlrc3-/- mice when treated with AOM/DSS, which possibly accounts for the hypersensitivity to carcinogenesis. Next, to further determine whether bone marrow- derived hematopoietic cells or stromal cells play a dominant role in the carcinogenesis of the Nlrc3-/- mice, Karki et al.13 established bone marrow chimeras and found that radioresistant stromal cells were more important than hematopoietic cells Figure 1 Functions of NLRs in inflammation-associated carcinogenesis. Under sustained during carcinogenesis, because cancer progression stimulation from pathogens, host cells or environmental sources, NLRs sense the was faster in Nlrc3-/- mice that received bone intracellular changes caused by infiltrating extracellular molecules or activation of the marrow from WT mice than in WT mice that downstream pathways of other PRRs and work to preserve cytosolic sanctity. NLRs function received bone marrow from Nlrc3-/- mice. The by associating with other proteins and through many interacting adaptors; however, details results from conditional knockout mice further are still unclear. NLRs can form inflammasomes to release IL-18 and IL-1β, which are pro- indicated that Nlrc3-/-intestinalepithelialcellswere inflammatory cytokines and related to repair and regeneration. In addition, NLRs can critical during carcinogenesis. Immunohistochem- regulate NF-κB, STAT3, MAPK, and AKT signaling pathways in an inflammasome- ical assessment of PCNA and Ki67 in colon tissues in vitro independent manner. Overwhelming sustained stimulation will dysregulate the expression of and an culture of colonic epithelial stem NLRs, which have unknown mechanisms. Karki et al.13 recently reported that NLRC3 is cells both demonstrated the high proliferation ability of colon epithelial cells when Nlrc3 was downregulated in AOM/DSS-induced colorectal tumor tissue, and the ability of NLRC3 to conditionally knocked out. Then, Karki et al.13 found associate with PI3K and inhibit PI3K/mTOR is impaired, which results in aberrant fl that the PI3K/mTOR pathway was aberrantly acti- proliferation of intestinal epithelial cells. Dysregulated NLRs will boost in ammation by Nlrc3 fl fl vated in the -/- epithelial cells. Activation of the promoting the production of pro-in ammatory cytokines and chemokines and then in uence PI3K/mTOR pathway has been shown to occur on the function of epithelial cells and immune cells, which favors a pro-tumor environment. day 8 in this model, which is prior to the increase in DAMP, danger-associated molecular pattern; IL-18, interleukin-18; MDSC, myeloid-derived inflammatory cytokines and immune cells. Co- suppressing cell; NLR, nucleotide-binding and oligomerization domain-like receptor; PAMP, immunoprecipitation experiments showed that pathogen-associated molecular pattern; PRR, pattern recognition receptor; TADC, tumor- NLRC3 could bind to PI3K to reduce the activity associated dendritic cell; TAM, tumor-associated macrophage; TLR, Toll-like receptor. of p85 and disrupt the association between the PI3K p85 and p110α subunits. All three domains of

Cellular & Molecular Immunology NLR members in carcinogenesis H Zhu and X Cao

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NLRC3wereshowntoparticipateinthisinteraction. same or similar signaling pathways. NLRP12, 8 Bruchard M, Rebe C, Derangere V, Togbé D, These results indicate that NLRC3 protects epithelial NLRP6, NLRP2 and NLRP4 can inhibit the NF- Ryffel B, Boidot R et al. The receptor cells from aberrant proliferation by interacting with κB pathway, whereas NOD1 and NOD2 promote NLRP3 is a transcriptional regulator of the PI3K/mTOR pathway and reducing the role of the NF-κBpathway.3 Although the knowledge of TH2 differentiation. Nat Immunol 2015; 16: 859–870. inflammation in colonic carcinogenesis. the sensing and regulatory mechanisms of NLRs has 9 Allam R, Maillard MH, Tardivel A, Chennupati V, The study by Karki et al.13 illustrates an essential 20–22 expanded in the most recent decade, the direct Bega H, Yu CW et al. Epithelial NAIPs protective role of NLRC3 in colonic epithelial cells interaction of NLRs with other proteins or ligands protect against colonic tumorigenesis. JExp during colitis-induced colorectal carcinogenesis and has not been extensively investigated. The study of Med 2015; 212:369–383. expands our understanding of functional mechan- Karki et al.13 is a good first step toward characteriz- 10 Ratsimandresy RA, Indramohan M, Dorfleutner A, isms of NLRC3 (Figure 1). A recent paper pub- fl ing these interactions. Regarding the complex and Stehlik C. The AIM2 in ammasome is a lished in the Journal of Experimental Medicine has central regulator of intestinal homeostasis sophisticated tumor–host communication, the revealed that another NLR member, NLR family through the IL-18/IL-22/STAT3 pathway. inflammatory regulatory mechanism in tumor- apoptosis inhibitory proteins (NAIPs), protects the Cell Mol Immunol 2017; 14: 127–142. associated immune cells and stromal cells may be colonic epithelium against tumorigenesis. Unlike 11 Sharma N, Jha S. NLR-regulated pathways NLRC3, NAIP knockout mice exhibit a decreased different from those in acute infection mediated by in cancer: opportunities and obstacles for inflammatory response despite the increased color- viruses and bacteria. More efforts are needed to therapeutic interventions. Cell Mol Life Sci 2016; 73:1741–1764. ectal carcinogenesis. NAIP knockout mice exhibit determine the function of NLRs in tumor- associated immune cells, stromal cells and cancer 12 Ahn H, Kang SG, Yoon SI, Kim PH, Kim D, activation of the STAT3 pathway and increased Lee GS. Poly-gamma-glutamic acid from cells. The functions of the NLRs in different stages anti-apoptotic and proliferation-related gene Bacillus subtilis upregulates pro-inflammatory fl expression in the colonic epithelial cells. Interest- of in ammation-associated carcinogenesis have cytokines while inhibiting NLRP3, NLRC4 ingly, NAIPs regulate the STAT3 pathway in an been further characterized by more investigations. and AIM2 inflammasome activation. Cell inflammasome-independent manner, which is In addition to the AOM/DSS model, which imitates Mol Immunol 2016; 13:1–9. inconsistent with the traditional view that NAIPs ulcerative colitis-associated colorectal cancer, other 13 Karki R, Man SM, Malireddi RK, Kesavard- form an inflammasome with NLRC4.9 In addition, inflammation-associated carcinogenesis should also hana S, Zhu Q, Burton AR et al. NLRC3 is an inhibitory sensor of PI3K-mTOR pathways NLRP6 has also been found to be expressed in be investigated, such as hepatitis-related hepatocel- in cancer. Nature 2016; 540:583–587. colonic epithelial cells and can assist in protecting lular carcinoma. Exploring the interplay between fl 14 Conti BJ, Davis BK, Zhang J, O'connor W Jr, epithelial cells in an in ammasome-dependent NLRs or between other PRRs under different fl Williams KL, Ting JP. CATERPILLER 16.2 manner. Aberrant NLRP6 in ammasomes have contexts will provide insight into the functional (CLR16.2), a novel NBD/LRR family mem- been demonstrated to induce altered microbiota network in cancer. Resolution of these conflicting ber that negatively regulates T cell function. 17 and boost tumorigenesis. These studies indicate findings will help reveal the role of NLR members JBiolChem2005; 280:18375–18385. the critical roles of NLRs in regulating epithelial in inflammation-associated carcinogenesis and 15ZhangL,MoJ,SwansonKV,WenH, cells during inflammation-associated carcinogenesis Petrucelli A, Gregory SM et al. NLRC3, a identify new cancer therapeutic targets. and reveal the different regulatory patterns of the member of the NLR family of proteins, is a various NLRs co-existing in a distinct cell type. negative regulator of innate immune signal- However, it is unknown whether the functions of CONFLICT OF INTEREST ing induced by the DNA sensor STING. – these NLRs are redundant, or whether they are The authors declare no conflict of interest. Immunity 2014; 40:329 341. 16 Luo Y, Wu X, Ma Z, Tan W, Wang L, Na D differently stimulated by different ligands under et al. Expression of the novel adipokine various disease contexts. Additional studies are C1qTNF-related protein 4 (CTRP4) sup- needed to address these questions. presses colitis and colitis-associated color- fi et al.13 The ndings of Karki revealed the 1 Hanahan D, Weinberg RA. Hallmarks of ectal cancer in mice. Cell Mol Immunol importance of the cellular location of the NLRs. cancer: the next generation. 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